The action of protein Ser/Thr phosphatase-type-1 [PP1] is a major determinant of the phosphorylation state of hundreds of proteins in eukaryotic cells, governing processes as diverse as mitosis, motility and metabolism. PP1 is an ancient enzyme that is extraordinarily conserved, with baker's yeast GLC7 being functionally equivalent to the mammalian PP1catalytic subunit. Yeast genetics and mammalian cell biochemistry has shown that PP1 is essential for cell survival and its multiple functions are accomplished by distributing the catalytic subunit among multiple regulatory subunits that control enzymatic activity, substrate specificity and intracellular localization. There are at least five different inhibitor phosphoproteins that exert a further level of control over PP1 activity, with the oldest and most conserved being inhibitor-2 (Inh2). Inh2 has an essential homologue in yeast called GLC8 that is required for chromosome segregation. During the current term of this project a 2-hybrid screen with Inh2 discovered five proteins that also bind PP1, probably due to interaction bridged via GLC7(PP1). Prominent among these proteins is the cell cycle Nek2 kinase, which binds PP1 and is indirectly activated by Inh2 inhibition of the bound PP1. Thus, PP1 exhibits binding to regulatory subunits and to Inh2, both at the same time, which contradicts and requires reformation of the paradigm for PP1 regulation. This continuation proposes to: 1) Determine the structural requirements for assembly of Nek2, PP1 and Inh2 into a centrosome control complex, testing the hypothesis that the Nek2::PP1 dimer presents a preferential target for binding of Inh2; 2) Elucidate the biochemical steps in triggering activation of the centrosome signaling complex C-Nap1::Nek2::PP1 ::inh2, testing the hypothesis that Inh2 inhibits PP1 and tips the balance between the competing kinase and phosphatase (Nek2::PP1) acting on the bound centrosome protein C-Nap1; 3) Examine centrosome cohesion in living cells, testing the hypothesis that Inh2 is necessary for activation of the Nek kinase signaling complex, separation of centrosomes and progression through mitosis. The results of this research will provide new fundamental knowledge of mechanisms controlling centrosomes and cell cycle progression that is relevant to understanding human disease.